Thymine derivatives and quinazoline-dione derivatives for the inhibition of hsp27

ABSTRACT

The present invention relates to novel HSP27 inhibitors, in particular thymine derivatives according to general formula (VI), (VII) or (VII) and phenothiazine derivatives according to formula (V), and to their use as drugs for the selective inhibition of the heat shock protein HSP27 (HSPB1), in particular for use in the treatment of carcinomas or cystic fibrosis, said inhibitors having a particularly advantageous activity in the lower micromolar or sub-micromolar active ingredient concentration range with respect to HSP27.

The present invention relates to novel thymine derivatives and their useas medicinal products for the selective inhibition of the heat shockprotein HSP27 (HSPB1).

It is known that some types of cancer respond poorly to treatment withcytostatic agents from the start. Even in patients with types of cancerthat are actually easy to treat, however, therapy can fail after acertain period. Resistances, whereby the tumour cells become insensitiveto cytostatic agents, represent one possible reason. A number ofbiological processes are known which are behind this.

For example, it has been shown that the treatment of various tumour celllines with cytostatic agents leads to increased expression of cellularproteins, which are important for the protection and stabilizing of thecorrect folding of receptors, enzymes and cellular structural proteins.

Heat shock proteins are evolutionarily highly conserved proteins, whichare formed by the cell under sublethal stress conditions. Heat shockproteins are chaperones, with natural tasks including returning proteinsthat have been denatured by heat or chemicals to their functional stateor preventing the denaturation of proteins. Because of the way in whichthey function, heat shock proteins are overexpressed in many cancercells. In recent years, therefore, heat shock proteins have emerged aspromising target proteins for cancer therapy.

The heat shock protein HSP27 (HSPB1) is involved in many cellularprocesses, e.g. apoptosis (programmed cell death), DNA repair andrecombination. HSP27 is overexpressed in the cell in many differenttypes of cancer, e.g. prostate cancer, bowel cancer, liver cancer andbreast cancer, and affects the course of the disease. It has been foundthat increased expression of HSP27 is associated with increasedresistance to cytostatic agents, such as e.g. gemcitabine(2′,2′-difluorodeoxycytidine) or bortezomib (Velcade), in chemotherapy.Because of the negative effect of HSP27 on chemotherapy, HSP27 isconsidered to be a promising additional target in cancer therapy, inparticular for suppressing the development of chemoresistance.

One possible therapeutic approach is described in JP 2010 215669 A or US2012/294846 A1 and is based on suppressing the gene expression of HSP27by direct intervention in translation or transcription by means ofintracellular administration of antisense oligonucleotides ordouble-stranded small interfering (si) RNA. Here, the nucleotide-basedinhibitors bind to the DNA or RNA, thereby specifically preventing theformation of HSP27 in the cell.

In connection with this, WO 2013 114339 A1 discloses a combinationtherapy for the treatment of cancer based on the synergistic effect of anucleotide inhibitor for inhibiting the expression of HSP27 inconjunction with an EGFR (epidermal growth factor receptor) proteininhibitor, such as e.g. erlotinib, or antifolates such as pemetrexed.The nucleotide inhibitor is preferably an antisense oligonucleotide or adouble-stranded siRNA.

However, one disadvantage in the use of nucleotide-based inhibitors istheir excess of negative charges and the associated high polarity of theactive ingredient molecules, whereby their bioavailability issignificantly minimized. Even more problematic is the huge chemicalinstability of nucleotide-based inhibitors, in particular RNA-basedinhibitors.

A further disadvantage is that the transfection (=introduction offoreign oligonucleotides into eukaryotic cells) of the nucleotide-basedinhibitors is complex and in some cases of only low efficiency. Inaddition, short nucleotide-based inhibitors have the disadvantage of apotential immunogenic action, also acting e.g. by secretion onnon-transfected cells (e.g. macrophages) and thus inducing a strongimmune response in the organism, which is harmful for the patient.

An alternative approach to the use of nucleotide-based inhibitors isrepresented by low-molecular-weight organic compounds, since theseusually exhibit high stability in a biological system together with goodbioavailability. In the case of highly specific compounds in particular,the “off-target” effects typical of other methods (e.g. due to theotherwise necessary transfection) do not occur.

DE 10 2008 035 299 A1 describes a method according to which thelow-molecular-weight organic compound(E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) interacts directly with theprotein HSP27, inhibiting the functionality of the HSP27. As a weakHSP27 inhibitor, BVDU thus increases sensitivity to cytostatic-inducedapoptosis, so that pancreatic cancer patients obtain a survival benefitthrough oral administration of BVDU. Clinical studies with late-stagepancreatic cancer patients have shown that BVDU works safely andefficiently at an administered dose of 500 mg/day. However, at anincreased administered dose of 760 mg/day, BVDU proved disadvantageousfor low-weight patients.

The compound BVDU described above and derivatives thereof in DE 10 2008035 299 A1 are the only known low-molecular-weight inhibitors of HSP27.The existing active ingredients show considerable potential forimprovement in terms of binding affinity.

WO 2009/156182 A2 describes uracil derivatives of general formula I

which are employed together with a cytostatic agent to suppress orreduce the development of resistance in cytostatic treatment. WO2009/156182 A2 shows no methods for the experimental validation of thesecompounds for the assumptions made; for instance, no values for theinhibitory concentration or for active ingredient concentrations of theindividual compounds can be taken from the document.

There is therefore an enormous need to provide more efficient HSP27inhibitors for the selective and direct inhibition of the HSP27 proteinin the context of use in a multifactorial combination therapy, inparticular for cancer therapy.

The present invention is therefore based on the object of providingchemical compounds that inhibit the heat shock protein HSP27 asselectively and potently as possible, in particular for use in cancertherapy.

In particular, it is the object of the present invention to providechemical compounds which, compared with compounds known from the priorart, exhibit a significant retardation or suppression of the developmentof resistance in cytostatic treatment in cancer chemotherapy.

To achieve the object, purine derivatives according to general formula(I) or (II) are specified for use as medicinal products, in particularfor use for the treatment of carcinomas or mucoviscidosis:

wherein:

-   -   R_(n) is selected from hydrogen (H),

-   -   wherein        is the covalent linkage to general formula (I) or (II) and the        variables have the following meanings:    -   U is selected from oxygen (O), an optionally branched and/or        OH-functionalized C₁ to C₄ alkyl residue and optionally        substituted vinyl residue, in particular substituted with        halogen, particularly preferably F, Cl, I; U is preferably O or        an optionally OH-functionalized C₁ to C₄ alkyl residue,    -   W is an optionally substituted methylene (—CH₂—), ethylene        (—CH₂CH₂—), ethoxy (—O—CH₂CH₂—) or ethene-1,2-diyl residue        (—CH═CH—), and wherein the substituents are preferably selected        from —CH₃, —CH₂OH, —COOCH₃, —CH₂—(PO₃H)_(o)—OH with o=0, 1, 2 or        3, —CH₂-phosphonic acid,    -   V is selected from H, —OH, an amino acid -AA, wherein the amino        acid is in particular selected from the group of proteinogenic        amino acids and their corresponding β-AAs and wherein AA is        bound covalently to W via the carboxyl group, an optionally        substituted heterocycle, in particular a six-membered ring        nitrogen heterocycle, such as pyridine, diazine, piperidine or        piperazine, and wherein the substituents are preferably selected        from —OH and an optionally substituted and/or polycyclic aryl        residue, in particular phenyl, naphthalene, quinoline,        naphthyridine, and wherein the substituents on the aryl residue        are preferably selected from —CF₃, —F, —Cl, —I, —OH, —NH₂, ═O,        —COOH, —OCH₃, —R,    -   A₁ and A₂ independently of one another are —CH₂, —CHOR, —CHF— or        —CHOC(═O)R,    -   G is CH₂, —CH₂O— or O,    -   Y is O, S, NR, carboxyl, carbonyl or amide,    -   wherein R is an H or an optionally OH-functionalized C₁ to C₈        alkyl residue,    -   the substituent R_(y) is H, OH, an optionally substituted C₁ to        C₇ alkyl residue, an optionally substituted cyclic or polycyclic        aryl residue or an optionally polycyclic nitrogen heterocycle,        in particular an optionally substituted phenyl, naphthalene,        bisphenyl, phenanthrene, benzopyrene, pyridine, diazine,        triazole, piperidine, bipiperidine, piperazine, xanthene,        carbazole, 9,10-dihydrophenanthrene, phenothiazine,        triphenylmethane residue or a combination thereof, wherein the        substituents are selected from —F, —NH₂, R and the group of the        six-membered ring nitrogen heterocycle, in particular pyridine,        1,2-diazine, 1,3-diazine, piperidine, bipiperidine or piperazine        or a sequence of two to three of the above-mentioned        substituents bridged to one another in para-position.        Alternatively, R_(y) is —C═O or is selected from        —CR_(a)R_(b)R_(c) wherein R_(a), R_(b) and R_(c) are selected        independently of one another from H and cyclic residues.

The object of the invention is furthermore achieved by thyminederivatives of general formula (VI) or (VII):

wherein the variables have the following meanings:

-   -   the substituent A is —H, —CH₃ or an optionally substituted C₂ to        C₄ vinyl or alkynyl residue, in particular substituted with        halogen, particularly preferably F, Cl, I; alternatively, A is a        halogen, —NO₂, —C═O, phenyl or thiophene residue—the halogen        preferably being selected from I and Br;    -   the linker L preferably serves to restrict the conformational        flexibility (i.e. reduction in entropy) and is selected from an        optionally substituted phenyl, benzyl or pyridine residue, in        particular substituted with —CH₃, —CF₃, halogen, particularly        preferably substituted with —CF₃ and F,

wherein

-   -   is the covalent linkage to general formula (VI) or (VII) or is        Y,    -   the substituents B₁ and B₂ independently of one another are —H,        —CH₃, —CF₃, —F or —Cl,    -   A₁, is —CH₂—, —CHOR, —CHF— or —CHOC(═O)R,    -   A₃ is —CH₂—, —CHOR, —CHF—, —CHOC(═O)R or —CHK—, wherein K is an        optionally substituted five-membered ring nitrogen heterocycle,        in particular triazole, diazole or imidazole, wherein the        five-membered ring nitrogen heterocycle is preferably bound        covalently to A₃ via a nitrogen atom.    -   G is CH₂, —CH₂O— or O;    -   Alternatively, the linker L is selected from optionally        substituted C1 to C6 alkyl residues or

wherein G and A₁ are selected as above and A₄ is —CH₂— or —CHOR and T isCH and/or T and G or G and A₄ together form a cyclopropyl ring;

-   -   Y is O, S, NR, carboxyl, carbonyl or amide,    -   wherein R is an H or an optionally substituted and/or branched        C₁ to C₈ alkyl residue,    -   the substituent R_(y) is as defined above or below H, OH, an        optionally substituted cyclic or polycyclic aryl residue or an        optionally substituted oxygen or nitrogen heterocycle, wherein        the substituents are selected from —F, —NH2, R and the group of        the six-membered ring nitrogen heterocycles, in particular        pyridine, 1,2-diazine, 1,3-diazine, piperidine, bipiperidine or        piperazine or a sequence of two to three of the above-mentioned        substituents bridged to one another in para-position;    -   Alternatively, R_(y) is —C═O or is selected from        —CR_(a)R_(b)R_(c) wherein R_(a), R_(b) and R_(c) are selected        independently of one another from H and cyclic residues,        preferably selected from optionally substituted cyclic or        polycyclic aryl residues and optionally substituted        heterocycles, particularly preferably H, phenyl, naphthylene,        biphenyl. Preferably, at least one of the residues R_(a), R_(b)        and R_(c) is H and at least one of the residues is a cyclic        residue, which is preferably selected as above;    -   the substituent R_(N) in formula (IV) is H or a benzoyl residue.

Advantageously, taking account of the steric effects, the substituentR_(y) on the purine derivative according to formula (I) or (II) or thethymine derivative of formula (VI), (VI′), (VII) or (VIII) is adapted tothe active ingredient binding pocket in such a way that the substituentR_(y) represents a planar system. The planar system does not necessarilyhave to be a conjugated system here. Preferably, the substituent R_(y)on the purine derivative according to formula (I) or (II) or the thyminederivative of formula (VI), (VI′), (VII) or (VIII) is an optionallysubstituted cyclic or polycyclic aryl residue or a nitrogen heterocycle,in particular selected from optionally substituted phenyl, naphthalene,bisphenyl, phenanthrene, benzopyrene, pyridine, diazine, xanthene,carbazole, phenothiazine, triphenylmethane, piperidine, bipiperidine,piperazine residues and a combination thereof. Preferably, takingaccount of the steric effects, the substituents of the cyclic orpolycyclic aryl residue or of the nitrogen heterocycle have an and/or −Meffect, whereby the purine derivatives of formula (I) or (II) and thethymine derivatives of formula (VI), (VI′), (VII) or (VIII)advantageously have a higher binding affinity to the active ingredientbinding pocket of the HSP27 protein. Preferably, the substituents of thecyclic or polycyclic aryl residue or of the nitrogen heterocycle areselected from -halogen, —NO₂, —CN, —N(R)₂, —SR, —OR, —COOR, —COR, R,wherein R as defined above is H or a C1 to C8 alkyl residue.Particularly preferably, the substituents of the cyclic or polycyclicaryl residue or of the nitrogen heterocycle are, independently of oneanother, substituents having an −I and/or −M effect selected from H,—NO₂, —CF₃, —F, —Cl, —Br, —OH, —COOH, —OCH₃ and —COR; quite particularlypreferably, the substituents are selected independently of one anotherfrom H, —NO₂, —CF₃, —OH, —COOH and F.

Alternatively or in addition, K is preferably selected independently ofR_(y) from the same residues as mentioned above for R_(y). This appliesin particular when R_(y) is H or OH. The purine derivative according toformula (I) or (II), in addition to the basic purine structure,preferably has at least one optionally substituted cyclic or polycyclicaryl residue or an optionally substituted nitrogen heterocycle in R_(n)or R_(y).

The thymine derivative according to formula (VI), (VI′), (VII) or (VIII)preferably has, in addition to the basic thymine structure, at least oneoptionally substituted aliphatic cyclic or polycyclic residue(preferably in L) or an optionally substituted cyclic or polycyclic arylresidue or an or an optionally substituted oxygen or nitrogenheterocycle (preferably in K or R_(y)). Preferably, the nitrogenheterocycle is further substituted. Particularly preferably, thenitrogen heterocycle is a triazole ring, which is preferably linked inposition 1 to Y or —CH in in —CHK and is preferably substituted inposition 4 of the triazole ring. Preferred substituents have the formula-R_(T)E with R_(T) selected from a single bond and C1 to C5 alkyl and Eselected from cyclic residues. The cyclic residues in E are preferablymol/Locyclic residues, particularly preferably selected fromcyclopentyl, pyridine and phenyl and mol/Lo- or polyhalogen (preferablyBr, Cl or F) substituted phenyl.

According to a particularly advantageous embodiment of the presentinvention, the linker L is an optionally substituted phenyl, benzyl orpyridine residue, in particular substituted with —CH₃, —CF₃ or halogen,particularly preferably substituted with —CF₃ and —F,

wherein the substituents B₁ and B₂ are, independently of one another,—H, —CH₃, —CF₃, —F or —Cl, A₁ and A₂ are, independently of one another,—CH₂—, —CHOR, —CHF— or —CHOC(═O)R and G is CH₂ or O. Thymine derivativesof general formula (VI), (VI′), (VII) or (VIII), in which the ligand isselected as listed above, exhibit a high binding affinity to the activeingredient binding pocket of the HSP27 protein (i.e. reduction inentropy).

Restriction of the conformational flexibility (i.e. reduction inentropy) within the meaning of the present invention means that acertain conformational flexibility is retained in the molecule, althoughthe number of possible conformations in the molecule and the energybarrier for the conformational flexibility of the molecule are severelyrestricted.

The term “optionally substituted” is understood to mean an unsubstitutedor substituted residue.

The term “polycyclic aryl residue” within the meaning of this inventionis to be understood as a conjugated system containing at least twoarylene groups which are connected together directly, i.e. are inconjugation with one another. The arylene groups here can also beconnected together directly via an electron donor (e.g. nitrogen,oxygen, CH) and thus create the planarity of the system, such as e.g.xanthenes, carbazoles, phenothiazines or triphenylmethane.

Advantageously, purine derivatives according to general formula (I) or(II) and thymine derivatives of formula (VI), (VI′), (VII) or (VIII)inhibit the HSP27 protein by means of a specific interaction with theactive ingredient binding pocket of the HSP27 protein.

The advantage of the purine derivatives according to general formula (I)or (II) and the thymine derivatives according to general formula (VI),(VI′), (VII) or (VIII) for use in cancer therapy consists in the factthat these compounds have an activity at least 50 times higher thancurrently known compounds for the selective inhibition of HSP27.

Particularly advantageously, the purine derivatives of general formula(I) or (II) and thymine derivatives according to general formula (VI),(VI′), (VII) or (VIII) already have an activity with respect to HSP27 inthe lower micromolar or submicromolar active ingredient concentrationrange. According to a preferred embodiment of the present invention, thepurine derivatives of general formula (I) and (II) and thyminederivatives of formula (VI), (VI′), (VII) or (VIII) or thyminederivatives of formula (VI), (VI′), (VII) and (VIII) are employed withan active ingredient concentration in the range of 1 nmol/L to 1000μmol/L, preferably in the range of 10 nmol/L to 750 μmol/L, furtherpreferably 100 nmol/L to 500 μmol/L or 10 nmol/L to 100 ρmol/L,particularly preferably 100 nmol/L to 10 μmol/L.

“Inhibition of HSP27” within the meaning of the present invention meansthat, by interaction with a low-molecular-weight compound, preferablythe purine derivative of general formula (I) or (II), the thyminederivatives according to general formula (VI), (VI′), (VII) or (VIII) orphenothiazine derivatives according to formula (V), the HSP27 loses itscapacity to interact with cancer-triggering binding partners (e.g.proteins) and at the same time advantageously induces the introductionof apoptosis (e.g. by stimulated activity of caspases). Caspases are themost important enzymes in apoptosis (programmed cell death) in animals.

The present invention is based on the very recent finding that HSP27,which is overexpressed in many types of cancer and has a negative effecton the course of the disease, has an active ingredient binding pocketfor low-molecular-weight organic compounds, said binding pocket beingcentral to the functionality (e.g. binding to client proteins andprobably also oligomerization) of the HSP27 protein and its activity.The functionality of the active ingredient binding pocket is based ontwo phenylalanine residues at positions 29 and 33 (Phe29 and Phe33) inthe amino acid sequence of the protein. Thus, it has been shown thatmutants lacking the corresponding phenylalanine residues are incapableof interacting with purine derivatives of general formula (I) or (II).

Since the corresponding crystal structural data for the HSP27 protein donot exist, the active ingredient binding pocket of HSP27 wascomputer-modelled with the aid of various in silico methods.

Within the meaning of the invention, the active ingredient bindingpocket of the HSP27 protein is a localized region (local tertiarystructure), which is formed by spatial arrangement of the amino acidsequence and comprises the phenylalanine residues Phe29 and Phe33 (basedon the primary structure of HSP27). The active ingredient binding pocketof HSP27 is capable of interacting with a low-molecular-weight organiccompound, preferably by means of coordinative interaction, in such a waythat the HSP27 protein loses its functionality (capacity foroligomerization and interaction with client proteins). A client proteinhere is a substrate, preferably a protein, on which HSP27 acts as achaperone. The binding affinity here is a measure of the bindingstrength between binding partners (here the low-molecular-weight organiccompound) and a target protein. The higher the binding affinity betweenthe binding partner and the target protein, the lower is thedissociation constant (K_(D)). The inhibitory concentration (K_(i)) ofthe binding partner refers to the concentration at which completeinhibition of the target protein is present. It is understood that thelower the value of K_(i), the higher the binding affinity.

Through a precise knowledge of the tertiary structure of the activeingredient binding pocket of HSP27, virtual screening can be used topre-identify low-molecular-weight organic compounds which, compared withknown compounds, interact significantly more specifically with theactive ingredient binding pocket of HSP27.

The term “virtual screening” comprises a medicinal product researchprocess known to a person skilled in the art, in which novel activeingredient molecules are to be found by means of in silico methods (i.e.in computer experiments). The binding affinity (K_(i)) of thesepotential inhibitors is not measured by wet chemical experiments here,as in traditional screening, but is predicted by computer-based methods.

By means of the binding/interaction of a low-molecular-weight organiccompound or inhibitor on the active ingredient binding pocket of HSP27,the structure of the HSP27 protein can undergo changes. Often, thesechanges are limited to side chain conformations, but entire groups ofamino acids with their peptide backbone can also move.

Advantageously, as a result of virtual screening, only a fewlow-molecular-weight organic compounds then have to be testedexperimentally by in vitro (i.e. outside a living organism), in situ(i.e. in cells, preferably in cell cultures) and/or in vivo (i.e. in theliving organism) experiments for their physicochemical affinity towardsthe active ingredient binding pocket of HSP27, which increasesefficiency in identifying specific HSP27 inhibitors and at the same timereduces costs. These in vitro experiments for identifying affinitytowards the active ingredient binding pocket of HSP27 take place e.g. bymeans of binding assays, in which the association of two unlabelledbinding partners is measured (e.g. bio-layer interferometry). In thecase of HSP27, furthermore, the function of the target protein or itsinhibition can be quantitatively measured in aggregation assays.

In in vitro experiments, it has now been found that the purinederivatives of general formula (I) or (II) and thymine derivatives offormula (VI), (VI′), (VII) or (VIII) according to the invention have adissociation constant (K_(D)) in respect of the HSP27 protein in therange of 10 nM/L to 1000 μmol/L, preferably in the range of 100 nM/L to800 μmol/L.

In situ, the inhibiting action of the pre-identifiedlow-molecular-weight organic compounds on the functionality of the HSP27protein is preferably measured in cells that express HSP27. Preferably,the in situ methods are performed with the aid of commol/L, readilycultured cell lines in which the binding partners are expressed.Suitable cell lines are U937 and RPMI-8226 as examples of cancer celllines and CCD-186Sk as an example of a mucoviscidosis cell line. Thedetection of the efficacy of the low-molecular-weight organic compoundspreferably takes place here by measuring the development of resistanceto a cytostatic agent or determining the interaction between HSP27 andits binding partners (e.g. by measuring the caspase-3 activity).

The term “low-molecular-weight organic compound” within the meaning ofthe invention is understood to mean molecular compounds composedprimarily of the elements carbon, hydrogen, oxygen and nitrogen andpreferably having a molecular weight in the range of 100 to 900 g/mol,in particular of 150 to 750 g/mol, above all 200 to 600 g/mol.

Low-molecular-weight organic compounds have the significant advantageover large molecules (e.g. nucleotide-based inhibitors such as antisenseoligonucleotides or RNA oligonucleotides) that they are substantiallyeasier to handle and more stable under physiological conditions.Furthermore, low-molecular-weight organic compounds are usuallycell-penetrating and therefore easier to administer into the cell or anorganism.

Surprisingly, it has now been found that in particular purinederivatives according to general formula (I) or (II) and thyminederivatives according to general formula (VI), (VI′), (VII) or (VIII) aslow-molecular-weight organic compounds bind selectively to the activeingredient binding pocket of the HSP27 protein and thus already inhibitthe functionality of the HSP27 protein in the lower micromolar orsubmicromolar concentration range.

Preferably, the substituent R_(y) is an optionally substituted cyclic orpolycyclic aryl residue or an optionally polycyclic and/or substitutednitrogen heterocycle, which is selected in particular from a phenyl,naphthalene, bisphenyl, phenanthrene, benzopyrene, pyridine, diazine,triazole, piperidine, bipiperidine, piperazine, xanthene, carbazole,phenothiazine, 9,10-dihydrophenanthrene, triphenylmethyl residue or acombinations thereof. Preferably, the six-membered ring nitrogenheterocycle is bound covalently to the general formula (I) or (II) via anitrogen atom.

According to an alternatively preferred embodiment of the presentinvention, the substituent R_(y) on the thymine derivative according togeneral formula (VI), (VI% (VII) or (VIII) is selected from a phenyl,triazole, piperazine, diazine and triphenylmethyl residue, which can inparticular be substituted with a six-membered ring nitrogen heterocycle,in particular pyridine, 1,2-diazine, 1,3-diazine, piperidine,bipiperidine or piperazine or a sequence of two to three of theabove-mentioned substituents bridged to one another in thepara-position.

It may optionally be provided that the substituent R_(y) on the thyminederivative according to general formula (VI) or (VII) is H or OH, i.e. asterically undemanding residue, wherein A₃ is particularly preferably—CHK—, i.e. a sterically demanding residue, wherein K is an optionallysubstituted five-membered ring nitrogen heterocycle, in particulartriazole, diazole or imidazole, wherein the five-membered ring nitrogenheterocycle is preferably bound covalently to A₃ via a nitrogen atom.The substituent on the five-membered ring nitrogen heterocycle here isselected in particular from a phenyl, pyridine and diazine residue.

According to a preferred embodiment of the present invention, thesubstituent R_(y) on the purine derivative according to general formula(I) or (II) or the thymine derivative according to general formula (VI),(VI′), (VII) or (VIII) is adapted to the active ingredient bindingpocket in such a way that R_(y) is selected from H, general formula(III) or formula (IV):

wherein:

-   -   is the covalent linkage to general formula (I), (II) or (VI),        (VI′), (VII) or (VIII),    -   X is N or CH,    -   Z is a single bond, CH₂, O, C(═O), S or NR_(x), in particular        CH₂, O, C(═O), S or NR_(x), particularly preferably CH₂, O, S or        NR_(x),    -   R_(x) is an optionally substituted and/or branched C₁ to C₄        alkyl residue,    -   R¹, R², R³ and R⁴ independently of one another are —H, -halogen,        —NO₂, —CN, —NR₂, and —SR, —OR, —COOR, —COR, —R or an optionally        substituted C₁ to C₄ vinyl residue or optionally substituted        aryl residue, wherein R as defined above is H or a 0₁ to 0₈        alkyl residue.

Alternatively, R_(y) is preferably selected from —CR_(a)R_(b)R_(c)wherein R_(a), R_(b) and R_(c) are selected independently of one anotherfrom H and cyclic residues, preferably selected from optionallysubstituted cyclic or polycyclic aryl residues and optionallysubstituted heterocycles, particularly preferably H, phenyl,naphthylene, biphenyl. Preferably, at least one of the residues R_(a),R_(b) and R_(c) is H and at least one of the residues is a cyclicresidue, which is preferably selected as above.

By adapting the structures to the active ingredient binding pocket ofthe protein, the purine derivatives according to general formula (I) and(II) or thymine derivatives of formula (VI), (VI′), (VII) and (VIII)particularly advantageously exhibit an activity in the lower micromolaror submicromolar, particularly preferably in the nanomolar concentrationrange.

According to a preferred embodiment of the present invention, thesubstituents R¹ to R⁴, taking into account the steric effects, have an−I and/or −M effect and therefore the purine derivatives according togeneral formula (I) or (II) or thymine derivatives according to generalformula (VI), (VI′), (VII) or (VIII) advantageously have a high bindingaffinity to the active ingredient binding pocket of the HSP27 protein(i.e. low K_(i) and K_(D)). Preferably, the substituents R¹ to R⁴independently of one another are substituents having an −I and/or −Meffect selected from H, —NO₂, —CF₃, —F, —Cl, —Br, —OH, —COOH, —OCH₃ and—COR; particularly preferably, the substituents are selectedindependently of one another from H, —NO₂, —CF₃, —OH, —COOH and F.According to a quite particularly preferred embodiment of the presentinvention, exclusively two, particularly preferably exclusively one ofthe substituents R¹ to R⁴ have/has a substituent other than H.

According to an alternatively preferred embodiment of the presentinvention, one of the substituents R¹ to R⁴, quite particularlypreferably R³, is an optionally substituted aryl residue having a marked+M effect, preferably a phenyl residue or a six-membered ring nitrogenheterocycle selected from pyridine, pyridazine, pyrimidine or pyrazine.Quite particularly preferably, one of the substituents R¹ to R⁴ is anoptionally substituted six-membered ring nitrogen heterocycle.

The binding affinity of purine derivatives of general formula (I) and(II) to the active ingredient binding pocket of the HSP27 protein isdetermined inter alia by the nature of the substituent R_(n).Preferably, R_(n) is a sterically undemanding, small substituentselected from H, an optionally branched C₁-C₄ alkyl or ether residue,wherein V is H or OH, an optionally substituted five-membered ring orsix-membered ring oxygen heterocycle. In particular, purine derivativesof general formula (I) or (II) in which R_(n) is H, an optionallyOH-functionalized methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, tert-butyl residue or an optionally substituted five-memberedring oxygen heterocycle substituted via C1′, in particular furan,ribose, deoxyribose or dideoxyribose, displayed a particularly highbinding affinity to the active ingredient binding pocket of the HSP27protein (i.e. low K_(i) and K_(D)). Preferably, such purine derivativesof general formula (I) and (II) in which R_(n) is as defined above havea K_(i) with respect to HSP27 in the range of 10 nmol/L to 5 mmol/L,particularly preferably in the range of 50 nmol/L to 5 mmol/L.

Alternatively, it can be provided that the substituent R_(n) issubstantially composed of two parts, wherein part 1 comprises U and Wand part 2 comprises V. Preferably, part 1 (U and W) is an optionallybranched C₁-C₄ alkyl or ether residue and part 2 (V) is a substitutedsix-membered ring nitrogen heterocycle, such as pyridine, diazine,piperidine or piperazine, and wherein the substituents of thesix-membered ring nitrogen heterocycle are preferably selected from —OHand an optionally substituted and/or polycyclic aryl residue, inparticular phenyl, naphthalene, quinoline, naphthyridine, and whereinthe substituents on the aryl residue are preferably selected from —CF3,—F, —OH, —NH₂, ═O, —COOH, —OCH₃ and a C1 to C3 alkyl residue.Preferably, such purine derivatives of general formula (I) and (II) inwhich R_(n) is as defined above have a K_(i) with respect to HSP27 atleast in the lower micromolar or submicromolar range, particularlypreferably in the nanomolar range, quite particularly preferably in therange of 10 nmol/L to 200 nmol/L.

According to a preferred embodiment of the present invention, thesubstituent R_(n) is

wherein V is an amino acid AA which is bound covalently to W via itscarboxyl group. Preferably, the amino acid is selected from the group ofproteinogenic amino acids and their corresponding β-AA, but AA is quiteparticularly preferably selected from the group of hydrophobic(═non-polar) proteinogenic amino acids and their corresponding β-AA,e.g. alanine, valine, leucine, isoleucine, phenylalanine or tryptophan,as a result of which the binding affinity of purine derivatives ofgeneral formula (I) and (II) to the active ingredient binding pocket ofHSP27 is advantageously further increased.

In particular, purine derivatives according to general formula (I) or(II) or thymine derivatives according to general formula (VI), (VI′),(VII) or (VIII) are preferred in which, for the substituent R_(y)according to formula (III), Z is —O—, —C(═O)— or —S— and X is N or CH,wherein purine derivatives in which Z is —O—or —C(═O)— and X is CH haveproved to be particularly selective HSP27 inhibitors, i.e. with highbinding affinity to the active ingredient binding pocket of the HSP27protein (i.e. low K_(i) and K_(D)).

Purine derivatives according to general formula (I) or (II) or thyminederivatives of formula (VI), (VI′), (VII) or (VIII) are preferred inwhich Y is an O, NR, a six-membered ring nitrogen heterocycle, inparticular pyridine, diazine, piperidine or piperazine, or an amidegroup (—NH—C(═O)—), wherein Y is particularly preferably an NR orpyridine, diazine, piperidine or piperazine. In the event that Y is anamide group, the amide group is preferably oriented such that the C(═O)group binds directly to the substituent R_(y) according to generalformula (III) or (IV).

According to a particularly preferred embodiment of the invention, R¹,R³ and optionally R⁴ of the substituent R_(y) on purine derivativesaccording to general formula (I) or (II) or thymine derivativesaccording to general formula (VI) are H. In this case, in particularvariations of purine derivatives according to general formula (I), (II)and thymine derivatives according to general formula (VI), (VI′), (VII)or (VIII) in which the phenyl ring according to formula (IV) onlycomprises the substituent R² in meta-position and wherein R¹ and R³ areH displayed particularly high binding affinity to the active ingredientbinding pocket of the HSP27 protein. According to a quite particularlypreferred embodiment of the present invention, for the substituent R_(y)according to formula (IV), R¹ and R³ are H and R² is an optionallysubstituted phenyl residue.

Alternatively, purine derivatives according to general formula (I) and(II) or thymine derivatives according to general formula (VI), (VI′),(VII) and (VIII) preferably only comprise the substituent R¹ inortho-position on the phenyl ring according to formula (IV) and/or R³ inpara-position, wherein R² in meta-position is H. According to a quiteparticularly preferred embodiment of the present invention, for thesubstituent R according to formula (IV), R¹ and R² are H and R³ is anoptionally substituted phenyl residue.

According to a quite particularly preferred embodiment of the presentinvention, for the substituent R_(y) according to formula (IV), R¹ is Hand R² and R³ are, independently of one another, R or an optionallysubstituted C₁ to 0₄ vinyl residue, wherein R is H or an optionallyOH-functionalized C₁ to C₅ alkyl residue.

According to a quite particularly preferred embodiment of the presentinvention, for the substituent R_(y) according to formula (III), R¹, R³and R⁴ are H and R² is H, -halogen, —COR or an optionally substitutedphenyl residue.

Also a subject of the invention are HSP27 inhibitors, in particularselected from the group of the purine derivatives of general formula (I)or (II), the thymine derivatives according to general formula (VI)and/or phenothiazine derivatives of general formula (V), for thesuppression of the development of chemoresistance in cancer therapy andfor use in the treatment of mucoviscidosis.

Mucoviscidosis is a hereditary disease, wherein in most cases of cysticfibrosis the CFTR protein (cystic fibrosis transmembrane conductanceregulator) at position 508, owing to a deletion of three nucleotides inthe DNA sequence coding for CFTR, lacks the amino acid phenylalanine(Phe508). The CFTR protein is a transmembrane protein, which regulatesthe transport of water and salt in the plasma membrane of epithelialcells. The resulting protein of the deletion mutant ΔF508 is not foldedentirely correctly during its formation (translation). It is thereforenot transported to the cell membrane via the endoplasmic reticulum, butis encapsulated with the assistance of HSP27 and transported fordegradation.

Commercially known treatment methods for mucoviscidosis only combat thevarious symptoms that are associated with the disease.

Surprisingly, it has now been found that, by means of the inhibition ofthe HSP27 protein by the specific interaction of the active ingredientbinding pocket of HSP27 with low-molecular-weight compounds, inparticular with purine derivatives of general formula (I) or (II),thymine derivatives according to general formula (VI), (VI′), (VII) or(VIII) and/or phenothiazine derivatives of general formula (V), thedeletion mutant ΔF508 is protected from degradation and consequently, toa certain degree, transported to the cell membrane where it isfunctionally integrated. Advantageously, the deletion mutant ΔF508 thathas been functionally integrated into the cell membrane can perform itsnatural function as a cAMP-regulated chloride channel.

According to a further preferred embodiment of the present invention, inparticular the purine derivatives of general formula (I) and (II) orthymine derivatives according to general formula (VI), (VI′), (VII) and(VIII) but also phenothiazine derivatives of general formula (V) aresuitable as medicinal products for use in cancer therapy, in particularfor suppressing the development of chemoresistance, and in the treatmentof mucoviscidosis. Also a subject of the invention is the use of thesecompounds for preparing a medicinal product, preferably for use incancer therapy, in particular for suppressing the development ofchemoresistance, and in the treatment of mucoviscidosis.

The present invention also comprises compounds such as pharmacologicallyacceptable salts, prodrugs, enantiomers, diastereomers, racemicmixtures, crystalline forms, amorphous forms and solvates, having apurine derivative according to general formula (I) and (II) or a thyminederivative of general formula (VI), (VI′), (VII) and (VIII), for use asmedicinal products in cancer therapy, in particular for suppressing thedevelopment of chemoresistance, and the treatment of mucoviscidosis.

A pharmacologically acceptable salt within the meaning of the presentinvention is understood to be chemical compounds of the purinederivative according to general formula (I) and (II) or of the thyminederivative of formula (VI), (VI′), (VII) and (VIII) composed ofpositively and negatively charged ions, which, depending on thesubstituents of the purine derivative, can be obtained e.g. by treatmentwith a weak, pharmaceutically acceptable acid or base.

The term prodrug refers to an inactive or less active pharmacologicalsubstance, which is only converted to the pharmacologically active formof a purine derivative according to general formula (I) or (II) or of athymine derivative according to general formula (VI), (VI′), (VII) and(VIII) by a chemical modification under physiological conditions (i.e.by metabolic processes or metabolization) in the organism. It can alsobe provided here that the prodrug is converted to the pharmacologicallyactive form of a purine derivative according to general formula (I),(II) or of a thymine derivative according to general formula (VI),(VI′), (VII) or (VIII) by means of chemical or biochemical methods in anex vivo environment.

The possibility of administering a compound containing a purinederivative according to general formula (I) or (II) and/or a thyminederivative according to general formula (VI), (VI′), (VII) or (VIII)into an organism depends on the structure of the compound. Aparticularly advantageous property of the purine derivatives accordingto general formula (I) or (II) and the thymine derivatives according togeneral formula (VI), (VI′), (VII) or (VIII) compared with conventionalnucleotide-based inhibitors is that, as low-molecular-weight organiccompounds within the meaning of the invention, they have abioavailability which is e.g. largely independent of the pH in thestomach. According to a particularly preferred embodiment of the presentinvention, compounds containing a purine derivative according to generalformula (I) and/or (II) and/or a thymine derivative of formula (VI) or(VII) are administered to an organism by an oral or parenteral route andare resorbed via the mucous membranes. For administration orally,pharmaceutical formulations e.g. in the form of a tablet, capsule orliquid, or rectally in the form of suppositories, are suitable. Examplesof organisms within the meaning of the present invention are preferablyselected from the group of the mammals, wherein the purine derivativeaccording to general formula (I) or (II) and/or thymine derivative offormula (VI), (VI′), (VII) or (VIII) can quite particularly preferablybe administered to a human.

A further subject of the invention is a pharmaceutical formulationcontaining at least one purine derivative according to general formula(I) or (II) and/or thymine derivatives according to general formula(VI), (VI′), (VII) or (VIII) for use in cancer therapy or the treatmentof mucoviscidosis.

In this case, the purine derivatives according to general formula (I) or(II) and/or the thymine derivatives according to general formula (VI),(VI′), (VII) or (VIII) can be used in the pharmaceutical formulation asthe sole pharmacologically active ingredient or in combination with atleast one cytostatic agent, e.g. in order to extend the spectrum ofactivity or prevent the development of resistances. In many cases,additive or synergistic effects are obtained here, i.e. the efficacy ofthe mixture is greater than the efficacy of the individual components.

It is known that the reduced activity of the HSP27 protein in the cell,independently of the administration of at least one cytostatic agent,already leads to a state of long-term stability in the malignantdegeneration of cancer cells and thus contributes to significantlyreduced tumour growth [Straume et al., 2012]. Against this background,according to a preferred embodiment of the present invention thepharmaceutical formulation according to the invention only has oneactive ingredient in the form of the purine derivative according togeneral formula (I) or (II) or the thymine derivative of general formula(VI) or (VII).

Particularly preferred, however, is a pharmaceutical formulationaccording to the invention containing the purine derivative according togeneral formula (I) or (II) and/or the thymine derivative of generalformula (VI), (VI′), (VII) or (VIII) in combination with at least onecytostatic agent. Such a pharmaceutical formulation according to theinvention thus offers the particular advantage that the development ofresistance during cytostatic treatment is prevented or at leastsignificantly delayed. Examples of cytostatic agents are: folic acidantagonists (e.g. methotrexate, pemetrexed), pyrimidine analogs (e.g.5-fluorouracil, gemcitabine), purine analogs (e.g. pentostatin,azathioprine) and oligopeptides with N- or C-terminal protection (e.g.bortezomib).

As described above, the purine derivative of general formula (I) or (II)and/or the thymine derivative of general formula (VI), (VI′), (VII) or(VIII) can be administered orally in the form of tablets, capsules,liquids or syrup or rectally in the form of suppositories.

It has also surprisingly been found that phenothiazine derivativesaccording to general formula (V):

wherein:

-   -   X is N and Z is S,    -   R_(x) is an optionally substituted and/or branched C₁ to C₄        alkyl, alkenyl or alkynyl residue,    -   the substituents R¹ and R² in ortho-, meta- or para-position        are, independently of one another, —H, —OR, —C(═O)R, —CF₃,        -halogen, an aliphatic or aromatic heterocycle,    -   Y is N or an amide group (—NR—C(═O)—),    -   n and m are, independently of one another, an integer from 0 to        3,    -   R⁵ and R⁶ are, independently of one another, H, an optionally        substituted piperazine, phenyl (—O₆H₅), hydroxyphenyl (—OC₆H₅)        or phenylene residue (—C₆H₄—),

are also capable of selectively binding to the active ingredient bindingpocket of the HSP27 protein, thereby inhibiting the functionality of theHSP27 protein, and so phenothiazine derivatives are suitable for use incancer therapy or the treatment of mucoviscidosis.

Typical examples of phenothiazine derivatives according to generalformula (V) are known to a person skilled in the art and are e.g.(MW=molecular weight):

Phenothiazine CAS Number derivatives Molecular formula MW [g/mol]61-00-7 Acepromazine C₁₉H₂₂N₂OS 326.46 50-53-3 ChlorpromazineC₁₇H₁₉ClN₂S 318.86 60-99-1 Levomepromazine C₁₉H₂₄N₂OS 328.47 58-40-2Promazine C₁₇H₂₀N₂S 284.42 60-87-7 Promethazine C₁₇H₂₀N₂S 284.42146-54-3 Triflupromazine C₁₈H₁₉F₃N₂S 352.42 84-97-9 Perazine C₂₀H₂₅N₃S339.50 58-39-9 Perphenazine C₂₁H₂₆ClN₃OS 403.97 69-23-8 FluphenazineC₂₂H₂₆F₃N₃OS 437.52

Alternative examples of phenothiazine derivatives according to generalformula (V) are also (with the respective calculated inhibitoryconcentration in relation to HSP27 -K_(i)):

Phenothiazine derivatives according to general formula (V) are employedin pharmacy in a known manner as antipsychotics, preferably in aconcentration of 1.0 to 3.0 mg per kg body weight of an organism.

Depending on the type of carcinoma to be treated, the combination of apurine derivative according to general formula (I) or (II) and/or athymine derivative of general formula (VI), (VI′), (VII) or (VIII) and aphenothiazine derivative according to general formula (V) canadvantageously lead to superadditive (“synergistic”) effects in theselective inhibition of the HSP27 protein. Thus, for example, thefollowing effects are possible, which go beyond the effects that wouldactually be expected: reduced administration concentration and/orextended spectrum of activity and/or increased efficacy of theindividual compounds (i.e. active ingredients) and pharmaceuticalformulations. In a preferred embodiment of the present invention, thepharmaceutical formulation according to the invention accordinglycomprises the purine derivative according to general formula (I) or (II)and/or the thymine derivative of general formula (VI), (VI′), (VII) or(VIII) in combination with one or more phenothiazine derivativesaccording to general formula (V).

Chlorpromazine displayed only a low affinity for interaction with HSP27in wet-chemical experiments and is therefore less preferred as arepresentative of the phenothiazine derivatives of general formula (V)for use according to the invention in cancer therapy or the treatment ofmucoviscidosis.

According to a preferred embodiment of the present invention,phenothiazine derivatives of general formula (V) are used individuallyor as a mixture thereof as medicinal products for use in cancer therapyor the treatment of mucoviscidosis.

A subject of the invention is therefore also the use of thephenothiazine derivatives of general formula (V) or of a mixture of thistype for the preparation of a pharmaceutical formulation for use incancer therapy or the treatment of mucoviscidosis.

Preferably, the phenothiazine derivatives of general formula (V) areadministered in a quantity of 0.1 to 50 mg/kg, preferably about 0.1 to30 mg/kg body weight per day and ideally in a quantity of 0.5 to 20mg/kg, particularly preferably 0.5 to 15 mg/kg or 0.3 μmol/kg to 150μmol/kg and ideally 1.5 μmol/kg to 60 μmol/kg body weight per day. Aperson skilled in the art will recognize that, if another compound isemployed, e.g. in combination with a purine derivative of generalformula (I) or (II) and/or a thymine derivative of general formula (VI),(VI′), (VII) or (VIII), the correct dosage can be determined both byinvestigating the efficacy of the compound in cell proliferation assaysand by establishing the toxicity in animal experiments (and ultimatelyin humans).

Furthermore, a method for reducing the functionality of the HSP27protein in a cell or organism is also covered by the present invention,comprising the administration of a purine derivative according togeneral formula (I) or (II) or a thymine derivative of general formula(VI), (VI′), (VII) or (VIII) in a physiologically effectiveconcentration for the inhibition of the HSP27 protein, wherein thepurine derivative interacts with the active ingredient binding pocket ofthe HSP27 protein.

The invention also comprises the use of a purine derivative according togeneral formula (I), (II) or a thymine derivative of general formula(VI), (VI′), (VII) or (VIII) and/or phenothiazine derivatives of generalformula (V) for the treatment of disease states which are associatedwith increased HSP27 signalling, in particular in cancer and also inmucoviscidosis.

The invention also comprises a method for the treatment ofmucoviscidosis or avoidance of the development of chemoresistance incancer therapy by administration of an effective dose of an HSP27inhibitor, in particular of a purine derivative according to generalformula (I), (II) or of a thymine derivative of general formula (VI) or(VII) (VI), (VI′), (VII) or (VIII) and/or phenothiazine derivatives ofgeneral formula (V).

The treatment method here comprises the administration of apharmaceutical formulation containing at least a purine derivativeaccording to general formula (I), (II), a thymine derivative of formula(VI), (VI′), (VII) or (VIII) and/or a phenothiazine derivative ofgeneral formula (V) in a physiologically effective concentration.According to a preferred embodiment of the invention, in particular forthe avoidance of the development of chemoresistance in cancer therapyand for the treatment of mucoviscidosis, the pharmaceutical formulationfor administration is solid or liquid, e.g. in the form of a tablet,capsule or liquid or rectally in the form of suppositories, so that thepharmaceutical formulation is administered to an organism by oral orparenteral administration and resorbed via the mucous membranes.

Preferably, the purine derivatives according to general formula (I),(II), thymine derivatives of formula (VI), (VI′), (VII) or (VIII) and/ora phenothiazine derivative of general formula (V) are administered in aquantity of about 0.1 to 30 mg/kg body weight per day and ideally in aquantity of 0.5 to 15 mg/kg body weight per day. Preferred are doses of0.1 to 500 μmol, in particular 0.5 to 200 μmol, particularly preferably1 to 50 μmol per kg body weight per day. Suitable organisms for suchtreatment are e.g. mammals, such as humans. Preferably, the purinederivatives according to general formula (I), (II), thymine derivativesof formula (VI), (VI′), (VII) or (VIII) and/or a phenothiazinederivatives of general formula (V) are employed in non-cytotoxicconcentrations.

A subject of the invention are also purine derivatives according togeneral formula (I) and/or (II), of a thymine derivative of formula(VI), (VI′), (VII) or (VIII) and/or of a phenothiazine derivative ofgeneral formula (V) or of a mixture for use as medicinal products, inparticular for use in cancer therapy or the treatment of mucoviscidosis.

According to a preferred embodiment of the present invention, therefore,the purine derivatives according to general formula (I) and/or (II),thymine derivatives of formula (VI), (VI′), (VII) or (VIII) and/orphenothiazine derivatives of general formula (V) or of a mixture areused for the preparation of a pharmaceutical formulation, in particularfor use in cancer therapy or the treatment of mucoviscidosis.

The dosage of the phenothiazine derivatives of general formula (V)and/or the purine derivatives of general formula (I) or (II) and/or thethymine derivatives of formula (VI), (VI′), (VII) or (VIII) depends onvarious factors, e.g. the administration method, age, weight and health,including the type of the organism to be treated.

Typical examples of purine derivatives according to general formula (I)or (II) (with the respective calculated inhibitory concentration inrelation to HSP27 - K_(i)) are:

The compounds with the lowest possible K_(i) are particularly preferred.Preferred are the compounds with a K_(i) of no more than 100 μmol/L, inparticular no more than 10 μmol/L, particularly preferably no more than500 nmol/L.

Typical examples of thymine derivatives according to general formula(VI), (VI′), (VII) or (VIII): (with the respective calculated inhibitoryconcentration in relation to HSP27 - K_(i)):

The compounds with the lowest possible K_(i) are particularly preferred.Preferred are the compounds with a K_(i) of no more than 100 μmol/L, inparticular no more than 10 μmol/L, particularly preferably no more than500 nmol/L.

The present invention also comprises the use of a purine derivativeaccording to general formula (I) and/or (II), a thymine derivative offormula (VI), (VI′), (VII) or (VIII) and/or a phenothiazine derivativeof general formula (V) for the preparation of a pharmaceuticalformulation, in particular for use in cancer therapy or the treatment ofmucoviscidosis.

A subject of the invention is also the use of a purine derivativeaccording to general formula (I), (II), a thymine derivative of formula(VI), (VI′), (VII) or (VIII) and/or a phenothiazine derivative ofgeneral formula (V) in combination with chemotherapy, radiotherapyand/or cancer immunotherapy for use in the treatment of cancer diseases,wherein advantageously the development of resistance (i.e. ability of atumour or tumour cells to resist the treatment method employed) to thetreatment methods is reduced, in particular suppressed.

Traditional methods of treating cancer diseases are e.g. surgical tumourremoval (resection), chemotherapy and radiotherapy, wherein often two oreven all three treatment methods are used at the same time in anorganism. In cancer immunotherapy methods, a distinction is made betweenactive and passive immunisation. In the case of active immunisation, thepatient is given substances which are intended to trigger an immuneresponse in his/her immune system. In the case of passive immunisation,antibodies or antibody fragments are employed. In adoptiveimmunotherapy, leucocytes are taken from the patient, cultured ex vivoand then injected into the patient again. However, if all the cells ofthe tumour and its metastases are not destroyed during the treatment,the further treatment of cancer diseases is made significantly moredifficult owing to the development of resistance.

A purine derivative according to general formula (I), (II), of a thyminederivative of formula (VI), (VI′), (VII) or (VIII) and/or of aphenothiazine derivative of general formula (V) therefore offers theparticular advantage that the development of resistance in chemotherapy,radiotherapy or cancer immunotherapy, in particular cytostatictreatment, is prevented or at least significantly delayed. Examples ofcytostatic agents are: folic acid antagonists (e.g. methotrexate,pemetrexed), pyrimidine analogs (e.g. 5-fluorouracil, gemcitabine),purine analogs (e.g. pentostatin, azathioprine) and oligopeptides withN- or C-terminal protection (e.g. bortezomib).

Preferably, at least one purine derivative according to general formula(I), (II), a thymine derivative of formula (VI), (VI′), (VII) or (VIII)and/or a phenothiazine derivative of general formula (V) is employed incombination with a cytostatic agent, which is preferably selected fromthe above-mentioned list, for the cancer therapy.

In a preferred therapy regimen, the purine derivative, thyminederivative and/or phenothiazine derivative is already employed beforethe start of the chemotherapy, radiotherapy and/or cancer immunotherapyand the administration of the purine derivative, thymine derivativeand/or phenothiazine derivative is continued during the said cytotoxictherapies (chemotherapy, radiotherapy and/or cancer immunotherapy) inorder to suppress the development of resistance. Preferably, theadministration of the purine derivative, thymine derivative and/orphenothiazine derivative is started 15 min to 4 hours before the startof the cytotoxic therapy.

Further features and advantages of the invention can be taken from thefollowing schematic drawings and embodiment examples with the aid ofwhich the invention is intended to be explained in more detail by way ofexample without limiting the invention thereto.

There are shown in:

FIG. 1: a spectrophotometric protein aggregation assay at 43° C., awavelength of 500 nm and a concentrations of the proteins: 1.44 μmol/Lcitrate synthase (CS), 481 nmol/L HSP27 (HSP) in a 40 mmol/L HEPESbuffer (pH 7.4) during treatment with 14 or 28 μmol/L2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one (CS+HSP+testsubstance no. 2), with 750 μmol/L BVDU (CS+HSP+BVDU) and without(CS+HSP; reference);

FIG. 2: the cell counts over the number of treatment days of U937 cellstreated with the cytostatic agent bortezomib (0.1 to 0.5 ng/ml) in eachcase combined with the HSP27 inhibitors acepromazine (+1 μM ACE) andBVDU (+30 μM BVDU) and without HSP27 inhibitor.

FIG. 3: the cell counts over the number of treatment days of U937 cellstreated with the cytostatic agent bortezomib (0.2 to 0.4 ng/ml) in eachcase combined with the HSP27 inhibitors acepromazine (+1 μM ACE) andBVDU (+30 μM BVDU) and without HSP27 inhibitor.

FIG. 4 shows the inhibition of the development of resistance of thetumour cell line RPMI-8226 by acepromazine. FIG. 4a shows RPMI 8226cells treated with cytostatic agent bortezomib 0.2-0.4 ng/ml (A),combination treatment with bortezomib plus 0.75 μM acepromazine (B) andcombination treatment with bortezomib plus 30 μM BVDU (C). FIG. 4b showsthe control without cytostatic agent: RPMI 8226 cells untreated (A),0.75 μM acepromazine alone (B) and 30 μM BVDU alone (C).

FIG. 5 shows the inhibition of the development of resistance of thetumour cell line RPMI-8226 by chlorpromazine. FIG. 5a shows RPMI 8226cells treated with cytostatic agent bortezomib 0.2-0.4 ng/ml (A),combination treatment with bortezomib plus 0.5 μM chlorpromazine (B) andcombination treatment with bortezomib plus 30 μM BVDU (C). FIG. 5b showsthe control without cytostatic agent: RPMI 8226 cells untreated (A), 0.5μM chlorpromazine alone (B) and 30 μM BVDU alone (C).

FIG. 6 shows the inhibition of the development of resistance of thetumour cell line RPMI-8226 by2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one.FIG. 6a shows RPMI 8226 cells treated with cytostatic agent bortezomib0.1-0.3 ng/ml (A), combination treatment with bortezomib plus 1 μM2-(4-butylphenylamino)-9-(2-hydroxy-ethoxymethyl)-1,9-dihydropurin-6-one(B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG.6b shows the control without cytostatic agent: RPMI 8226 cells untreated(A), 1 μM2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-onealone (B) and 30 μM BVDU alone (C).

FIG. 7 shows the inhibition of the development of resistance of thetumour cell line RPMI-8226 by2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one. FIG. 7a showsRPMI 8226 cells treated with cytostatic agent bortezomib 0.1-0.3 ng/ml(A), combination treatment with bortezomib plus 1 μM2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one (B) andcombination treatment with bortezomib plus 30 μM BVDU (C). FIG. 7b showsthe control without cytostatic agent: RPMI 8226 cells untreated (A), 1μM 2-(3-trichlorovinyl-phenylamino)-1,9-dihydropurin-6-one alone (B) and30 μM BVDU alone (C).

FIG. 8 shows the inhibition of the development of resistance of thetumour cell line RPMI-8226 by 9H-xanthene-9-carboxylic acid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide.FIG. 8a shows RPMI 8226 cells treated with cytostatic agent bortezomib0.075-0.275 ng/ml (A), combination treatment with bortezomib plus 1 μM9H-xanthene-9-carboxylic acid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide(B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG.8b shows the control without cytostatic agent: RPMI 8226 cells untreated(A), 1 μM 9H-xanthene-9-carboxylic acid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amidealone (B) and 30 μM BVDU alone (C).

FIG. 9 shows the inhibition of the development of resistance of thetumour cell line RPMI-8226 by2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide.FIG. 9a shows RPMI 8226 cells treated with cytostatic agent bortezomib0.075-0.275 ng/ml (A), combination treatment with bortezomib plus 1 μM2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]acetamide(B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG.9b shows the control without cytostatic agent: RPMI 8226 cells untreated(A), 1 μM2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]acetamidealone (B) and 30 μM BVDU alone (C).

FIG. 10 shows the inhibition of the development of resistance of thetumour cell line U-937 by acepromazine. FIG. 10a shows U-937 cellstreated with cytostatic agent bortezomib 0.1-0.5 ng/ml (A), combinationtreatment with bortezomib plus 0.75 μM acepromazine (B) and combinationtreatment with bortezomib plus 30 μM BVDU (C). FIG. 11b shows thecontrol without cytostatic agent: RPMI 8226 cells untreated (A), 0.75 μMacepromazine alone (B) and 30 μM BVDU alone (C).

FIG. 11 shows the inhibition of the development of resistance of thetumour cell line U-937 by2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one.FIG. 11a shows U-937 cells treated with cytostatic agent bortezomib0.1-0.5 ng/ml (A), combination treatment with bortezomib plus 0.75 μM2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one(B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG.11b shows the control without cytostatic agent: RPMI 8226 cellsuntreated (A), 0.75 μM2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-onealone (B) and 30 μM BVDU alone (C).

EXAMPLE 1 SpeedScreen of Purine Derivatives

For the experimental validation of low-molecular-weight compounds whichact as potential HSP27 inhibitors according to the in silico prediction,a method based on gel filtration was employed.

The method for detecting molecular bonds using gel filtration wasdescribed in 2004 as “SpeedScreen” [Muckenschnabel et al., 2004] and wasadapted to meet the requirements of the invention. The method ofseparation consists in the fact that larger molecules, such as e.g.proteins, can pass through a matrix of Sephadex G-25, whilelow-molecular-weight compounds (small molecules) penetrate into thematrix and do not pass through it. For the separation, PD SpinTraps G-25from GE Healthcare were used. During the 2-minute centrifugation at800×g (according to operating instructions), these columns allowmolecules with a molecular weight of greater than or equal to 5000 g/molto pass through, while smaller molecules are retained in the matrix.

The target protein HSP27 has a molecular weight of 27000 g/mol andtherefore passes through the matrix. The HSP27 inhibitors of generalformula (I) described according to the invention arelow-molecular-weight compounds, which can only pass through the SephadexG-25 matrix if they have previously bound to the target protein HSP27and are transported thereby through the matrix. Non-boundlow-molecular-weight compounds are retained by the matrix. Moleculesthat have passed through the matrix can then be detected by massspectroscopy. This enables “binders” to be differentiated from“non-binders”.

Result

Representatives of the purine derivatives according to general formula(I) tested by SpeedScreen are:

-   -   2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one    -   2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one    -   2-(3-hydroxymethylphenylamino)-3,7-dihydropurin-6-one

Furthermore, acepromazine (as acepromazine maleate) was testedpositively as a representative of the phenothiazine derivatives by meansof SpeedScreen.

The three purine derivatives pass through the Sephadex G-25 matrix andare detected in the medium after the passage. The binding of thephenothiazine derivatives acepromazine (as acepromazine maleate) andchlorpromazine to HSP27 could also be positively detected by means ofbio-layer interferometry with a K_(D)=72 μmol/L and 770 μmol/Lrespectively.

EXAMPLE 2 SpeedScreen of Thymine Derivatives

For the experimental validation of low-molecular-weight compounds whichact as potential HSP27 inhibitors according to the in silico prediction,a method based on gel filtration was employed.

The method for detecting molecular bonds using gel filtration wasdescribed in 2004 as “SpeedScreen” [Muckenschnabel et al., 2004] and wasadapted to meet the requirements of the invention. The method ofseparation consists in the fact that larger molecules, such as e.g.proteins, can pass through a matrix of Sephadex G-25, whilelow-molecular-weight compounds (small molecules) penetrate into thematrix and do not pass through it. For the separation, PD SpinTraps G-25from GE Healthcare were used. During the 2-minute centrifugation at800×g (according to operating instructions), these columns allowmolecules with a molecular weight of greater than or equal to 5000 g/molto pass through, while smaller molecules are retained in the matrix.

The target protein HSP27 has a molecular weight of 27000 g/mol andtherefore passes through the matrix. The HSP27 inhibitors of generalformula (I) described according to the invention arelow-molecular-weight compounds, which can only pass through the SephadexG-25 matrix if they have previously bound to the target protein HSP27and are transported thereby through the matrix. Non-boundlow-molecular-weight compounds are retained by the matrix. Moleculesthat have passed through the matrix can then be detected by massspectroscopy. This enables “binders” to be differentiated from“non-binders”.

Result

Representatives of the thymine derivatives according to general formula(VI) tested by SpeedScreen are:

-   -   9H-xanthene-9-carboxylic acid        [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide    -   2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide

Both thymine derivatives pass through the Sephadex G-25 matrix and aredetected in the medium after the passage.

The binding of the phenothiazine derivatives acepromazine (asacepromazine maleate) and chlorpromazine to HSP27 could also bepositively detected by means of bio-layer interferometry with a K_(D)=72μmol/L and 770 μmol/L respectively.

EXAMPLE 3 Detection of Binding by Bio-Layer Interferometry

Bio-layer interferometry is a technique for detecting biomolecularinteractions. It is an analytical technique that measures the opticalinterference of white light which is reflected by two surfaces: in eachcase, one surface consists of a layer of the immobilized target proteinon the tip of the biosensor and one is an internal reference surface.The binding of ligands to the target protein alters the interferencepattern and can be measured in real time.

For the measurements, an instrument of the ForteBio Octet Red 384 brandwas used. The target protein HSP27 was biotinylated and bound to theappropriate sensors (SSA). One great advantage of this analytical methodconsists in the fact that the analytes do not have to be eitherimmobilized or labelled. The purine derivatives of general formula (I)or (II) pre-identified by means of in silico screening are employed forthese measurements in a concentration range (K_(D)) of between 333μmol/L and 10 nmol/L.

Using the bio-layer interferometry method, the purine derivative2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-onecould be positively detected as an HSP27-binding low-molecular-weightcompound with a K_(D)=15 μmol/L.

The binding of the phenothiazine derivatives acepromazine (asacepromazine maleate) and chlorpromazine to HSP27 could also bepositively detected by means of bio-layer interferometry with a K_(D)=72μmol/L or 770 μmol/L respectively.

EXAMPLE 4 Spectrophotometric Protein Aggregation Assay

To measure the efficacy of the purine derivatives according to theinvention, robust experiments for the chaperone function of the HSP27protein were performed:

A known client protein (=substrate on which HSP27 acts as a chaperone)of HSP27 is citrate synthase. Citrate synthase is denatured when thetemperature increases to 43° C. This heat denaturation is prevented ordelayed by the presence of HSP27 [Jakob U, 1993]. By adding the novelactive ingredients, their efficacy can thus be measured through theinhibition of the HSP27 chaperone function.

To determine the activity (i.e. the effect on the aggregation behaviourof the citrate synthase (CS) after thermal incubation) of2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one in comparison toBVDU, four different batches containing 1.44 μmol/L CS, 481 nmol/L HSP27(HSP) in a 40 mmol/L HEPES buffer (pH 7.4) were prepared and BVDU (750μmol/L) or various concentrations of2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one (14 μmol/L; 28μmol/L) were added. The samples were then incubated at 43° C. and theaggregation behaviour of the CS was mol/Litored in a spectrometer(PerkinElmer LS55, PerkinElmer Inc.) at a wavelength of 500 nm.

It can be seen from FIG. 1 that the protein citrate synthase (graph CSalone) is denatured by increasing the temperature to 43° C. and formsaggregates. The aggregation is measured in the spectrometer at 500 nm(light scattering by protein aggregates). The presence of HSP27 (HSP)counteracts the aggregation (cf. graph, CS+HSP). Since the addition ofthe test substances (BVDU and2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one) inhibits thechaperone function of HSP27, the activity of the respective testsubstance can be determined by measuring the aggregation behaviour ofthe CS.

It is clear that CS alone forms aggregates very rapidly; these CSaggregates then precipitate in the measuring cuvette, so that the redgraph falls again after reaching the peak. Through the presence ofHSP27, CS aggregation is prevented over the period of one hour shownhere. BVDU partially cancels the chaperone function of HSP27 at aconcentration of 750 μmol/L BVDU. The test substance2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one shows acomparable effect at a concentration of 14 μmol/L and a significantlymore pronounced effect at 28 μmol/L.

The inhibition of HSP27 correlates to the binding strength of alow-molecular-weight organic compound to the active ingredient bindingpocket of HSP27. The active concentration of a low-molecular-weightorganic compound interacting with HSP27 can be converted to BVDUequivalents via the activity of BVDU, i.e. equal to the HSP27 inhibitingdose in mg BVDU. Based on 100 mg BVDU, the test substance2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one has a BVDUequivalent dose of less than 1.9 mg.

EXAMPLE 5 Capillary Electrophoresis of the CS Protein Precipitates withPurine Derivatives

Since the heat-denatured protein aggregates of the citrate synthase fromExample 1 are not water-soluble, these can be separated completely fromthe supernatant by means of centrifugation at room temperature at16,000×g, 10 min. By using capillary electrophoresis, the compositionsof the separated precipitates can be shown and quantified. The relativequantity of protein of precipitated citrate synthase serves as a measureof the efficacy of the test substances.

In each case, 481 nmol/L HSP27 and 1.44 μmol/L citrate synthase in 40mmol/L HEPES buffer, pH 7.4, were employed. The relative quantity ofprotein of the precipitated citrate synthase in each case was normalizedusing a defined quantity of BSA as internal standard. The relativequantity of protein that precipitated when using 750 μmol/L BVDU was setat 1. The other test substances (2 representatives of phenothiazinederivatives of general formula (V), 2 representatives of purinederivatives of formula (I) or (II)) were each employed at aconcentration of 10 μmol/L.

Table 1 below shows the separation by capillary electrophoresis ofheat-denatured protein aggregates of citrate synthase after thermaltreatment of the samples at 43° C. for 120 minutes.

Overview of test results: Rel. quantity Efficacy of protein comparedDose Substance employed prec. CS with BVDU 750 μmol/L  BVDU 1 10 μmol/LChlorpromazine 1.06 79.5 x 10 μmol/L Acepromazine 0.99  74.25 x 10μmol/L 2-(4-Butylphenylamino)-9-(2- 0.92 69.0 xhydroxyethoxymethyl)-1,9- dihydropurin-6-one 10 μmol/L2-(3-Trichlorovinyl- 0.71  53.25 x phenylamino)-1,9- dihydropurin-6-one

The four test substances proved significantly more effective than thereference substance BVDU when used at a concentration of 10 μmol/L ineach case, and can therefore be given in a dose in the range of between53 and 79 times lower to achieve an effect comparable with that of theconventional BVDU. In particular, the purine derivative2-(4-butyl-phenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-oneshows a 69 times higher activity than BVDU for the inhibition of theHSP27 protein (BVDU equivalent dose is 1.4 mg). However, the purinederivative 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one alsoshows a 53 times higher activity for the inhibition of the HSP27 proteincompared with BVDU, which corresponds to a BVDU equivalent dose of lessthan 1.9 mg.

EXAMPLE 6 Testing the HSP27 Inhibitors in Cancer Cells

Cancer cells develop resistances extremely rapidly when they are treatedwith cytostatic agents. This is the reason for numerous failures inchemotherapy. HSP27 favours the development of chemoresistances, e.g. byinteracting with apoptosis proteins and thus preventing the sought celldeath of the cancer cells. For bortezomib (Velcade) in particular, amodern cytostatic agent (proteasome inhibitor), the HSP27-drivendevelopment of resistance is well documented [Chauhan D, 2004].

In the cell experiments described here, it is shown that, byadministering the novel HSP27 inhibitors, the development of resistanceto the cytostatic agent bortezomib is prevented in U937 cells. In thiscase, acepromazine was tested in comparison with BVDU.

For this purpose, U937 cells (histiocytic lymphoma) were cultured inDMEM culture medium (+10% FCS) at 37° C., 5% CO₂ in an H₂O-saturatedatmosphere. 100,000 cells/ml were seeded and incubated with thecytostatic agent bortezomib (0.1 ng/ml) and the respective testsubstance (dosage in the current experiment: 1 μM/L acepromazine). Thereference substance, BVDU, was employed at a concentration of 30 μM/L.The cells were in each case passaged before the cell count reached1,000,000 cells/ml. At each passage, 100,000 cells/ml were again seededand the dose of the cytostatic agent bortezomib was increased stepwise(0.1 ng/mL or 0.05 ng/mL). The concentration of the test substancesremained constant. As a result of the stepwise increase in the dosage ofthe cytostatic agent, resistance developed in the U937 cells. Thedevelopment of resistance is prevented or inhibited in the case of apositively tested HSP27 inhibitor.

It can be seen from FIG. 2 that, by treating U937 cells withacepromazine (+1 μM ACE), no development of resistance occurred andtherefore no living cells were detectable after the end of theexperiment. In contrast, for U937 cells treated with BVDU (+30 μM BVDU),a final cell count of 430,000 cells/ml was detected whereas the finalcell count in the culture without the addition of an HSP27 inhibitoramounted to 790,000 cells/ml. As a control, U937 cells were treated withthe respective test substances alone. At the same dosage, the testsubstances alone have no effect on cell growth, since HSP27 is notneeded in a large quantity by the cancer cells without the pressure bythe cytostatic agent. The harmful effect of HSP27 for the patient on thedevelopment of resistance only becomes clear when the cytostatic agentis administered simultaneously.

EXAMPLE 7 Testing the HSP27 Inhibitors in Cancer Cells

The experiments from Example 6 were repeated in the same way, whereinU937 cells (histiocytic lymphoma) are cultured in DMEM culture medium(+10% FCS) at 37° C., 5% CO₂ in an H₂O- saturated atmosphere. 100,000cells/ml were seeded and incubated with an initial concentration of 0.2ng/ml of the cytostatic agent bortezomib and both without an inhibitorand with acepromazine as an HSP27 inhibitor at a concentration of 1μM/L. The reference substance, BVDU, was employed at a concentration of30 μM/L. The cells were passaged in each case before the cell countreached 1,000,000 cells/ml. At each passage, 100,000 cells/ml were againseeded and the dose of the cytostatic agent bortezomib was increasedstepwise by 0.1 ng/mL. The concentration of the test substance and thereference substance remained constant. As a result of the stepwiseincrease in the dosage of the cytostatic agent, resistance developed inthe U937 cells. The development of resistance is prevented or inhibitedin the case of a positively tested HSP27 inhibitor.

It can be seen from FIG. 3 that, by treating U937 cells withacepromazine (+1 μM ACE), no development of resistance occurred andtherefore no living cells were detectable after the end of theexperiment. In contrast, for U937 cells treated with BVDU (+30 μM BVDU),a final cell count of 60,000 cells/ml was detected whereas the finalcell count in the culture without the addition of an HSP27 inhibitoramounted to 230,000 cells/ml.

As a control, U937 cells were treated with the respective testsubstances alone. At the same dosage, the test substances alone have noeffect on cell growth, since HSP27 is not needed in a large quantity bythe cancer cells without the pressure by the cytostatic agent. Theharmful effect of HSP27 for the patient on the development of resistanceonly becomes clear when the cytostatic agent is administeredsimultaneously.

EXAMPLE 8 Capillary Electrophoresis of the CS Protein Precipitates withThymine Derivatives

Since the heat-denatured protein aggregates of the citrate synthase fromExample 2 are not water-soluble, these can be separated completely fromthe supernatant by means of centrifugation at room temperature at16,000×g, 10 min. By using capillary electrophoresis, the compositionsof the separated precipitates can be shown and quantified. The relativequantity of protein of precipitated citrate synthase serves as a measureof the efficacy of the test substances.

In each case, 481 nM HSP27 and 1.44 μmol/L citrate synthase in 40 mmol/LHEPES buffer, pH 7.4, were employed. The relative quantity of protein ofthe precipitated citrate synthase in each case was normalized using adefined quantity of BSA as internal standard. The relative quantity ofprotein that precipitated when using 750 μmol/L BVDU was set at 1. Theother test substances (2 representatives of phenothiazine derivatives ofgeneral formula (V), 2 representatives of thymine derivatives of formula(I) or (II)) were employed at a concentration of 10 μmol/L in each case.

Table 2 below shows the separation by capillary electrophoresis ofheat-denatured protein aggregates of citrate synthase after thermaltreatment of the samples at 43° C. for 120 minutes.

Overview of the test results: Rel. quantity Efficacy of protein comparedDose Substance employed prec. CS with BVDU 750 μmol/L  BVDU 1 10 μmol/LChlorpromazine 1.06 79.5 x  10 μmol/L Acepromazine 0.99 74.25 x 10μmol/L 9H-Xanthene-9-carboxylic 0.65 48.75 x acid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H- pyrimidin-1-yl)-but-2- enyl]amide 10 μmol/L2-Biphenyl-4-yl-N-[4-(5- 0.81 60.75 x methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)- but-2-enyl]acetamide

The four test substances proved significantly more effective than thereference substance BVDU when used at a concentration of 10 μmol/L ineach case, and can therefore be given in a dose in the range of between49 and 79 times lower to achieve an effect comparable with that of theconventional BVDU. In particular, the thymine derivative2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]acetamideshows a 61 times higher activity than BVDU for the inhibition of theHSP27 protein (BVDU equivalent dose is 1.6 mg). However, the thyminederivative 9H-xanthene-9-carboxylicacid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amidealso shows a 49 times higher activity for the inhibition of the HSP27protein compared with BVDU, which corresponds to a BVDU equivalent doseof less than 2 mg.

Further Test Results

Table 3 below shows the results of the capillary electrophoreticseparation of heat-denatured protein aggregates of citrate synthaseafter thermal treatment of the samples at 43° C. for 70 minutes forfurther thymine derivatives. Otherwise, the experiments were performedas described above:

Rel. quantity Efficacy of protein compared Dose Substance employed prec.CS with BVDU 750 μmol/L  BVDU 1 10 μmol/LN-[(2Z)-4-(2,4-Dioxo-3H-pyrimidin-1-yl)but-2-en-1-yl]-2- 1.15 86.25 x(naphthalen-2-yl)acetamide 10 μmol/LN-[(2Z)-4-(2,4-Dioxo-3H-pyrimidin-1-yl)but-2-en-1-yl]-2-(4- 0.88 66.15 xphenylphenyl)acetamide 10 μmol/LN-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.86 64.31 x1-yl]-9H-fluorene-9-carboxamide 10 μmol/L3′-Deoxy-3′-[4-(3,4-dichlorophenyl)-1H-1,2,3-triazol-1-yl]- 0.86 64.21 xthymidine 10 μmol/LN-[(2Z)-4-(2,4-Dioxo-3H-pyrimidin-1-yl)but-2-en-1-yl]-9,9a- 0.85 63.38 xdihydro-4aH-xanthene-9-carboxamide 10 μmol/L3′-Deoxy-3′-[4-(2-pyridinyl)-1H-1,2,3-triazol-1]-yl]thymidine 0.84 62.86x 10 μmol/L N-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en-0.83 62.51 x 1-yl]-2-(naphthalen-2-yl)acetamide 10 μmol/LN-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.82 61.19 x1-yl]-2,2-diphenylacetamide 10 μmol/L1-[(4S,5S)-4-[4-(3-Bromophenyl)-1H-1,2,3-triazol-1-yl]-5- 0.81 60.88 x(hydroxymethyl)oxolan-2-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione 10 μmol/LN-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.81 60.44 x1-yl]-2-(naphthalen-1-yl)acetamide 10 μmol/L3′-Deoxy-3′-(4-propyl-1H-1,2,3-triazol-1-yl)thymidine 0.79 59.59 x 10μmol/L 3′-Deoxy-3′-(4-phenyl-1H-1,2,3-triazol-1-yl)thymidine 0.79 59.07x 10 μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-2,2- 0.7958.97 x diphenylacetamide 10 μmol/LN-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.78 58.73 x1-yl]naphthalene-1-carboxamide 10 μmol/L3′-(4-Butyl-1H-1,2,3-triazol-1-yl)-3′-deoxythymidine 0.76 57.08 x 10μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-2- 0.75 56.36 x(naphthalen-2-yl)acetamide 10 μmol/L1-[(4S,5S)-5-(Hydroxymethyl)-4-[4-(4-methoxyphenyl)-1, 0.75 55.89 x2,3-triazol-1-yl]oxolan-2-yl]-5-methyl-3H-pyrimidine-2,4- dione 10μmol/L 3′-Deoxy-3′-[4-(2-phenylethyl)-1H-1,2,3-triazol-1-yl]- 0.74 55.67x thymidine 10 μmol/L3′-Deoxy-3′-(4-pentyl-1H-1,2,3-triazol-1-yl)thymidine 0.70 52.57 x 10μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-9H- 0.68 51.16x xanthene-9-carboxamide 10 μmol/L3′-[4-(4-Chlorophenyl)-1H-1,2,3-triazol-1-yl]-3′- 0.67 50.53 xdeoxythymidine 10 μmol/LN-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-2-(4- 0.67 50.52 xphenylphenyl)acetamide 10 μmol/L3′-Deoxy-3′-[4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl]- 0.66 49.69 xthymidine 10 μmol/L 3′-[4-(Cyclopentylmethyl)-1H-1,2,3-triazol-1-yl]-3′-0.66 49.63 x deoxythymidine 10 μmol/L1-[(4S,5S)-4-[4-(4-Bromophenyl)-1,2,3-triazol-1-yl]-5- 0.64 48.14 x(hydroxymethyl)oxolan-2-yl]-5-methyl-3H-pyrimidine-2,4- dione 10 μmol/L3′-Deoxy-3′-[4-(2-methyl-2-propanyl)-1H-1,2,3-triazol-1- 0.61 46.01 xyl]thymidine 10 μmol/LN-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-9H- 0.58 43.30 xfluorene-9-carboxamide

EXAMPLE 9 Treatment of Mucoviscidosis

To determine the effects on the concentration of deletion mutant AF508CFTR functionally integrated into the membrane, the mucoviscidosis cellline CCD-186Sk (ATCC® CRL-1563™) was used.

The mucoviscidosis cell line CCD-186Sk is a homozygous carrier of theAF508 mutation, to which the CFTR gene relates. The deletion of thephenylalanine at position 508 of the CFTR gene is typical of thisdisease and affects over 70% of patients. Because of this deletion, theCFTR protein that forms is not folded entirely correctly and istherefore supplied to the cellular degradation processes and not—likethe healthy protein—transported to the cell membrane and incorporatedthere as a transmembrane protein. HSP27 plays a crucial role inisolating the delta F508 CFTR protein for degradation. The experimentsserve to prove the hypothesis that delta F508 CFTR protein which is notisolated and degraded immediately upon its formation is transported tothe cell membrane where, as a functioning transmembrane protein, itregulates the transport of water and salt through the plasma membrane ofthe cells.

For this purpose, the CCD-186Sk cells are seeded at a density of 100,000cells/ml and incubated with:

-   -   2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one    -   2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one    -   2-(3-hydroxymethyl-phenylamino)-3,7-dihydropurin-6-one

as representatives of the purine derivatives in the respectiveconcentration. Every 24 hours, cells are harvested and tests areperformed to see whether the treated cells present more CFTR at the cellsurface than untreated cells. To detect the CFTR protein at the cellsurface, an antibody is used which specifically labels the extracellular“loop” (amino acids 103-117) of the CFTR protein. This antibody islabelled with the fluorescent dye FITC and detected by flow cytometry.

EXAMPLE 10 Bio-Layer Interferometry

For measurements by bio-layer interferometry, biotinylated HSP27 proteinwas first bound to Super-Streptavidin sensors (SSA, Fortebio). Theloaded sensors were then incubated (“quenched”) with biocytin. Asreference, unloaded SSA sensors were incubated with biocytin. In thecase of a specific binding to HSP27 of the analyte to be measured, theloaded HSP27 sensor generates a stronger signal (response) than theconcurrent reference sensor. After the “baseline” has been establishedby incubation with the reaction buffer (PBST 0.1% Tween 20 plus 3%DMSO), first the association and then the dissociation of the respectiveanalyte is measured in real time. The analytes were measured inincreasing concentrations in each case: 1.23-3.7-11.11-33.33-100-300μM/l.

Results:

Dissociation constant Substance K_(D) [μM] Chlorpromazine 770Acepromazine 72 5-Phenyl-2′-deoxyuridine 602-(3-Hydroxymethylphenylamino)-3,7-dihydropurin-6- 710 one2-(4-Butylphenylamino)-9-(2-hydroxyethoxymethyl)- 151,9-dihydropurin-6-one 9H-Xanthene-9-carboxylic acid [4-(5-methyl-2,4-267 dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide2-Biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro- 4242H-pyrimidin-1-yl)-but-2-enyl]acetamide

EXAMPLE 11 Inhibition of the Development of Resistance of the TumourCell Line RPMI-8226 by Acepromazine

The cell line RPMI-8226 (DMSZ 402) is a “multiple myeloma” cell line andwas chosen because bortezomib is employed as a standard therapy formultiple myeloma. RPMI-8226 cells respond to bortezomib, express HSP27and develop resistance to Velcade.

RPMI-8226 cells were seeded at a cell count of 100,000 cells/ml in eachcase and regularly passaged. FIG. 4a shows that, as a result of thetreatment with 0.75 μmol/L acepromazine, the development of resistanceto the cytostatic agent bortezomib was significantly inhibited and only30,000 living cells per ml medium could be detected at the end of theexperiment. In contrast, for RPMI 8226 cells that were treated with 30μmol/L BVDU, a final cell count of 160,000 cells/ml was detected whereasthe final cell count for the culture without the addition of an HSP27inhibitor amounted to 350,000 cells/ml. As a control, RPMI 8226 cellswere treated with the respective test substances alone, FIG. 4b . At thesame dosage, the test substances alone do not affect cell growth, sinceHSP27 is not needed in a large quantity by the cancer cells without thepressure from the cytostatic agent.

EXAMPLE 12 Inhibition of the Development of Resistance of the TumourCell Line RPMI-8226 by Chlorpromazine

RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml ineach case and regularly passaged. FIG. 5a shows that, as a result of thetreatment with 0.5 μmol/L chlorpromazine, the development of resistanceto the cytostatic agent bortezomib was significantly inhibited and only70,000 living cells per ml medium could be detected at the end of theexperiment. In contrast, for RPMI 8226 cells that were treated with 30μmol/L BVDU, a final cell count of 160,000 cells/ml was detected whereasthe final cell count in the culture without the addition of an HSP27inhibitor amounted to 350,000 cells/ml. As a control, RPMI 8226 cellswere treated with the respective test substances alone, FIG. 5b . At thesame dosage, the test substances alone do not affect cell growth.

EXAMPLE 13 Inhibition of the Development of Resistance of the TumourCell Line RPMI-8226 by2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one

RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml ineach case and regularly passaged. FIG. 6a shows that, as a result of thetreatment with 1 μM/L2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one,the development of resistance to the cytostatic agent bortezomib wassignificantly inhibited. At the end of the experiment, 240,000 livingcells per ml medium could be detected. For the treatment with 30 μM/LBVDU, a final cell count of 290,000 cells/ml was detected whereas thefinal cell count in the culture without the addition of an HSP27inhibitor amounted to 950,000 cells/ml. As a control, RPMI 8226 cellswere treated with the respective test substances alone, FIG. 6b . At thesame dosage, the test substances alone do not affect cell growth.

EXAMPLE 14 Inhibition of the Development of Resistance of the TumourCell Line RPMI-8226 by2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one

RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml ineach case and regularly passaged. FIG. 7a shows that, as a result of thetreatment with 1 μmol/L2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one, the developmentof resistance to the cytostatic agent bortezomib was significantlyinhibited. At the end of the experiment, 270,000 living cells per mlmedium could be detected. For the treatment with 30 μmol/L BVDU, a finalcell count of 290,000 cells/ml was detected whereas the final cell countin the culture without the addition of an HSP27 inhibitor amounted to950,000 cells/ml. As a control, RPMI 8226 cells were treated with therespective test substances alone, FIG. 7b . At the same dosage, the testsubstances alone do not affect cell growth.

EXAMPLE 15 Inhibition of the Development of Resistance of the TumourCell Line RPMI-8226 by 9H-xanthene-9-carboxylicacid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide

RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml ineach case and regularly passaged. FIG. 8a shows that, as a result of thetreatment with 1 μmol/L 9H-xanthene-9-carboxylicacid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide,the development of resistance to the cytostatic agent bortezomib wassignificantly inhibited. At the end of the experiment, 290,000 livingcells per ml medium could be detected. For the treatment with 30 μmol/LBVDU, a final cell count of 340,000 cells/ml was detected whereas thefinal cell count in the culture without the addition of an HSP27inhibitor amounted to 510,000 cells/ml. As a control, RPMI 8226 cellswere treated with the respective test substances alone, FIG. 8b . At thesame dosage, the test substances alone do not affect cell growth.

EXAMPLE 16 Inhibition of the Development of Resistance of the TumourCell Line RPMI-8226 by2-biphenyl-4-yl-N[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide

RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml ineach case and regularly passaged. FIG. 9a shows that, as a result of thetreatment with 1 μmol/L2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide,the development of resistance to the cytostatic agent bortezomib wassignificantly inhibited. At the end of the experiment, 380,000 livingcells per ml medium could be detected. For the treatment with 30 μmol/LBVDU, a final cell count of 340,000 cells/ml was detected whereas thefinal cell count in the culture without the addition of an HSP27inhibitor amounted to 510,000 cells/ml. As a control, RPMI 8226 cellswere treated with the respective test substances alone, FIG. 9b . At thesame dosage, the test substances alone do not affect cell growth.

EXAMPLE 17 Inhibition of the Development of Resistance of the TumourCell Line U-937 by Acepromazine

The cell line U-937 is a histiocytic lymphoma (DSMZ ACC 5). This cellline also responds to treatment with bortezomib, expresses HSP27 anddevelops resistance to bortezomib. Bortezomib is not the standardtherapy for this lymphoma, but is being discussed as a possibletherapeutic agent.

U-937 cells were seeded with a cell count of 100,000 cells/ml in eachcase and regularly passaged. FIG. 10a shows that, as a result of thetreatment with 0.75 μmol/L acepromazine, the development of resistanceto the cytostatic agent bortezomib was significantly inhibited. At theend of the experiment, 70,000 living cells per ml medium could bedetected. For the treatment with 30 μmol/L BVDU, a final cell count of250,000 cells/ml was detected whereas the final cell count in theculture without the addition of an HSP27 inhibitor amounted to 450,000cells/ml. As a control, U-937 cells were treated with the respectivetest substances alone, FIG. 10b . At the same dosage, the testsubstances alone do not affect cell growth.

EXAMPLE 18 Inhibition of the Development of Resistance of the TumourCell Line U-937 by2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one

U-937 cells were seeded with a cell count of 100,000 cells/ml in eachcase and regularly passaged. FIG. 11a shows that, as a result of thetreatment with 0.75 μmol/L2-(4-butyl-phenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one,the development of resistance to the cytostatic agent bortezomib wassignificantly inhibited. At the end of the experiment, 30,000 livingcells per ml medium could be detected. For the treatment with 30 μmol/LBVDU, a final cell count of 250,000 cells/ml was detected whereas thefinal cell count in the culture without the addition of an HSP27inhibitor amounted to 450,000 cells/ml. As a control, U-937 cells weretreated with the respective test substances alone, FIG. 11b . At thesame dosage, the test substances alone do not affect cell growth.

References

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Jakob U, Gaestel M, Engel K, and Buchner J. (1993) Small Heat ShockProteins Are Molecular Chaperones. J Biol Chem 268(3): 1517-1520.

Muckenschnabel I, Falchetto R, Mayr L M, Filipuzzi I. (2004)SpeedScreen: label-free liquid chromatography-mass spectrometry-basedhigh-throughput screening for the discovery of orphan protein ligands.Anal Biochem. 2004 Jan. 15; 324(2):241-9.

Chauhan D, Li G, Auclair D, Hideshima T, Podar K, Mitsiades N, MitsiadesC, Chen L B, Munshi N, Saxena S, Anderson K C. (2004) 2-Methoxyestardioland bortezomib/proteasome-inhibitor overcome dexamethasone-resistance inmultiple myeloma cells by modulating Heat Shock Protein-27. Apoptosis.2004 March; 9(2): 149-55.

1. Thymine derivative according to general formula (VI) for use asmedicinal product in the treatment of carcinomas or mucoviscidosis:

wherein: the substituent A is —H, halogen, —CH₃, —C═O, a C₂ to C₄ vinylresidue or an alkynyl residue, phenyl or a thiophene residue, the linkerL is an optionally substituted C1 to C6 alkyl residue, an optionallysubstituted phenyl, benzyl or pyridine residue,

wherein the substituents B₁ and B₂ independently of one another are —H,—CH₃, —CF₃, —F or —Cl, A₁ is —CH₂—, —CHOR, —CHF— or —CHOC(═O)R, A₃ is—CH₂—, —CHOR, —CHF—, —CHOC(═O)R or —CHK—, wherein K is a five-memberedring nitrogen heterocycle, A₄ is —CH₂— or —CHOR, G is —CH₂—, —CH₂O— or—O—, T is CH, and/or T and G or G and A₄ together form a cyclopropylring, Y is O, S, NR, carboxyl, carbonyl or amide, wherein R is H or a C₁to C₈ alkyl residue, the substituent R_(y) is H, OH, —C═O,—CR_(a)R_(b)R_(c), an optionally substituted cyclic or polycyclic arylresidue or an optionally substituted oxygen or nitrogen heterocycle,wherein R_(a), R_(b) and R_(c) independently of one another are selectedfrom H and cyclic residues, the substituent R_(N) is H or a benzoylresidue.
 2. Thymine derivative according to claim 1, characterized inthat the substituent R_(y) is selected from H, general formula (III) orformula (IV)

wherein:

is the covalent linkage to general formula (I) or (II), X is N or CH, Zis a single bond, CH₂, O, C(═O), S or NR_(x), R_(x) is an optionallysubstituted and/or branched C₁ to C₄ alkyl residue, R¹, R², R³ and R⁴each independently of one another are —H, -halogen, —NO₂, —CN, —NR₂ and—SR, —OR, —COOR, —COR, —R a C₁ to C₄ vinyl residue or an aryl residue,wherein R is H or C₁ to C₈ alkyl residue.
 3. Thymine derivativeaccording to claim 1, characterized in that the substituent R_(y) isselected from H and OH and A₃ is —CHK—, wherein K is a five-memberedring nitrogen heterocycle.
 4. Thymine derivative according to claim 3,characterized in that Z is O, C(═O) or S and X is N or CH.
 5. Thyminederivative according to claim 1, characterized in that Y is O, NR or anamide group.
 6. Thymine derivative according to claim 2, characterizedin that R¹, R³ and optionally R⁴ are H.
 7. Thymine derivative accordingto claim 2, characterized in that, for the substituent R_(y) accordingto formula (IV), R¹ is H, R² and R³ independently of one another are Ror an optionally substituted C₁ to C₄ vinyl residue, wherein R is H oran optionally OH-functionalized C₁ to C₅ alkyl residue.
 8. Thyminederivative according to claim 2, characterized in that, for thesubstituent R_(y) according to formula (III), R¹, R³ and R⁴ are H and R²is H, -halogen, —COR or an optionally substituted phenyl residue. 9.HSP27 inhibitors according to claim 1 for use in cancer therapy tosuppress the development of chemoresistance or in the treatment ofmucoviscidosis.
 10. A pharmaceutical formulation comprising at least onethymine derivative according to claim 1 in a physiologically effectiveconcentration for the treatment of carcinomas or mucoviscidosis.
 11. Thepharmaceutical formulation according to claim 10, further comprising oneor more phenothiazine derivatives according to formula (V):

wherein: X is N and Z is S, R_(x) is an optionally substituted and/orbranched C₁ to C₄ alkyl, alkenyl or alkynyl residue, the substituents R¹and R² in ortho-, meta- or para-position independently of one anotherare —H, —OR, —C(═O)R, —CF₃, -halogen, an aliphatic or aromaticheterocycle, Y is N or an amide group (—NR—C(═O)—), n and mindependently of one another are an integer from 0 to 3, R⁵ and R⁶independently of one another are H, a piperazine residue, a phenylresidue (—C₆H₅), a hydroxyphenyl residue (—OC₆H₅) or a phenylene residue(—C₆H₄—).
 12. (canceled)
 13. (canceled)
 14. A method for treating cancercomprising administering an pharmaceutically effective amount of athymine derivative according to claim 1 to a patient having cancer whois undergoing chemotherapy, radiotherapy and/or cancer immunotherapy.15. A method for treating cancer comprising administering apharmaceutically effective amount of the pharmaceutical formulationaccording to claim 10 to a patient having cancer who is undergoingchemotherapy, radiotherapy and/or cancer immunotherapy.
 16. A method fortreating cancer comprising administering a pharmaceutically effectiveamount of the pharmaceutical formulation according to claim 11 to apatient having cancer who is undergoing chemotherapy, radiotherapyand/or cancer immunotherapy.